Human metapneumovirus (HMPV) was first identified in the Netherlands in 2001 and soon after was isolated in patients with respiratory tract disease throughout the world, particularly in the pediatric population. HMPV replicates inefficiently in cell culture, posing a challenge to research. The contribution of HMPV to human disease remains to be defined but is approximately similar to that of human parainfluenza virus type 3, and thus there is a need for an HMPV vaccine, especially for the pediatric population. An HMPV vaccine likely would be a live-attenuated strain that would be given intranasally, likely in combination with live-attenuated vaccines that are being developed against human respiratory syncytial virus (HRSV) and the human parainfluenza viruses (HPIVs). HMPV is an enveloped virus with a genome that is a single negative-sense strand of RNA, and is classified in the paramyxovirus family together with HRSV and the HPIVs. We recently described the first complete sequence of the HMPV genome, and prepared complete consensus sequences for viruses (CAN97-83 and CAN97-75) representing the two genetic subgroups of HMPV (A and B, respectively). The HMPV genome sequenced to date range in length from 13,280-13,335 nt. The genome contains 8 genes that are in the order 3?-N-P-M-F-M2-SH-G-L-5? and have open reading frames corresponding to 9 major proteins. By analogy to HRSV, which has been studied in much greater detail, the HMPV proteins are: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; M2-1, RNA synthesis factor; M2-2, RNA synthesis factor; SH, small hydrophobic protein of unknown function; G, attachment glycoprotein; and L, viral polymerase. The HMPV proteins have only been deduced from the nucleotide sequence and had not been characterized directly with regard to their biochemistry or function. Compared to HRSV, HMPV lacks the non-structural NS1 and NS2 genes and has the F, M2, SH and G genes in the order F-M2-SH-G compared to SH-F-G-M2 for HRSV. The two HMPV subgroups share 81% nucleotide identity and 88% aggregate amino acid identity, similar to the respective values of 81% and 88% for the two HRSV subgroups. We developed a reverse genetic system for the CAN97-83 isolate, whereby changes can be introduced into the genome of infectious virus by recombinant DNA techniques. We designed a version of HMPV, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette placed 58 nt from the 3' end of the genome. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery and characterization of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals, and the ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. The rHMPV-GFP virus was used to develop a more rapid and reliable assay for HMPV-neutralizing antibodies We also recovered a version of HMPV without the added GFP gene. This virus replicated in vitro as efficiently as biologically-derived HMPV (showing that we had made a correct virus), whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold (showing that the addition of an extra gene was slightly inhibitory). Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in the promoter proximal position in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb in length, 30% longer than that of the natural virus. This rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes in order to increase gene dose or to provide representation of additional antigenic lineages or subgroups of HMPV. The ability to produce infectious HMPV from full-length cDNA provides a method to investigate the functions of individual HMPV proteins and to develop attenuating mutations for the purposes of constructing a live vaccine. As a first step, we engineered a HMPV to delete the SH or G genes individually or in combination. The del-SH, del-G, and del-SH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Indeed, the del-G virus grew marginally better, and the del-SH virus unambigously better, than their wild-type parent, whereas the double deletion mutant replicated marginally less efficiently. Thus, the SH and G proteins are not essential for efficient growth in cell culture. The SH, G and F proteins were identified for the first time by immunoprecipitation using peptide-specific sera. This showed that the SH protein accumulates in a variety of forms that range in apparent electrophoretic mobility from 23-220 kDa, with the differences appearing to be due to glycosylation. The G protein also appeared to be heavily glycosylated. Apart from the absence of the deleted protein(s), the virions produced by the gene-deletion mutants were very similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. This showed that neither SH nor G is essential for the efficient production of virus particles. However, subtle differences in yield and in sucrose sedimentation were noted that will be further investigated. When administered intranasally to hamsters, the del-SH virus replicated at least as efficiently as wild-type rHMPV. This indicates that SH is completely dispensable in vivo and that its deletion does not confer a significant attenuating effect, at least in this rodent model. The del-G and del-SH/G mutants also replicated in both the upper and lower respiratory tract, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were found to be strongly attenuated for replication in both the upper and lower respiratory tract (at least 600-fold and 40-fold reduction, respectively, of mean titer on day 3 post infection compared to wild-type rHMPV). The immunogenicity of the del-SH virus was comparable to that wild-type rHMPV, consistent with its high level of replication. Although the del-G and del-SH/G viruses were strongly attenuated, they also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Thus, the del-G and del-SH/G viruses represent promising vaccine candidates that will be prepared for clinical evaluation. Additional mutants were made involving the M2 gene, which encodes an mRNA with two overlapping ORFs that have the potential to encode two separate proteins M2-1 and M2-2. Expression of M2-1 was confirmed for the first time by immunoprecipitation with antiserum raised against HMPV, whereas expression of the M2-2 protein from recombinant HMPV was visualized by adding an epitope tag added to its carboxy-terminus. Recombinant HMPV were generated in which expression of M2-1 and M2-2 was silenced individually or together. This showed that neither protein is required for HMPV replication. These deletion viruses are being evaluated to characterize the effects of these deletions in vitro and in vivo and to determine the potential of these viruses as candidate vaccines.